Compounds and compositions as c-kit and PDGFR kinase inhibitors

ABSTRACT

The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with abnormal or deregulated kinase activity, particularly diseases or disorders that involve abnormal activation of c-kit, PDGFR and PDGFR kinases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 U.S. national phase application ofinternational application number PCT/US2008/062568 filed 2 May 2008,which application claims priority to U.S. provisional patent applicationNo. 60/916,051, filed 4 May 2007. The full disclosure of theseapplications is incorporated herein by reference in its entirety and forall purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention provides a novel class of compounds, pharmaceuticalcompositions comprising such compounds and methods of using suchcompounds to treat or prevent diseases or disorders associated withabnormal or deregulated kinase activity, particularly diseases ordisorders that involve abnormal activation of c-kit, PDGFRα and PDGFRβkinases.

2. Background

The protein kinases represent a large family of proteins, which play acentral role in the regulation of a wide variety of cellular processesand maintaining control over cellular function. A partial, non-limiting,list of these kinases include: receptor tyrosine kinases such asplatelet-derived growth factor receptor kinase (PDGF-R), the nervegrowth factor receptor, trkB, and the fibroblast growth factor receptor,FGFR3, B-RAF; non-receptor tyrosine kinases such Abl and the fusionkinase BCR-Abl, Lck, Bmx and c-src; and serine/threonine kinases such asc-RAF, sgk, MAP kinases (e.g., MKK4, MKK6, etc.) and SAPK2α and SAPK2β.Aberrant kinase activity has been observed in many disease statesincluding benign and malignant proliferative disorders as well asdiseases resulting from inappropriate activation of the immune andnervous systems.

The novel compounds of this invention inhibit the activity of one ormore protein kinases and are, therefore, expected to be useful in thetreatment of kinase-associated diseases.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds of Formula I:

L is selected from —NC(O)—, —NC(O)N— and —C(O)N—;

R₁, R_(2a) and R_(2b) are each independently selected from hydrogen,hydroxy, C₃₋₈heterocycloalkyl, C₁₋₄alkyl, C₁₋₄alkoxy,halo-substituted-C₁₋₄alkoxy, halo-substituted-C₁₋₄alkyl, —NR₁₀R₁₁,—OX₁R₈; wherein X₁ is selected from a bond and C₁₋₄alkylene; R₈ isC₃₋₁₂cycloalkyl; or R₁ and R_(2a) or R₁ and R_(2b) together with thecarbon atoms to which R₁ and R_(2a) or R_(2b) are attached form phenyl;R₁₀ and R₁₁ are independently selected from hydrogen, C₁₋₄alkyl,C₁₋₄alkoxy, halo-substituted-C₁₋₄alkoxy, halo-substituted-C₁₋₄alkyl,C₃₋₈heterocycloalkyl, C₁₋₁₀heteroaryl; or R₁₀ and R₁₁ together with thenitrogen to which R₁₀ and R₁₁ are both attached formC₃₋₈heterocycloalkyl or C₁₋₁₀heteroaryl;

R₃, R₄, R₅, R₆ and R₇ are independently selected from hydrogen, halo,cyano, C₁₋₆alkyl, C₁₋₆alkoxy, halo-substituted-C₁₋₆alkoxy,C₃₋₈heterocycloalkyl, —OX₂R₉, —S(O)₀₋₂R₉ and —NR₁₂R₁₃; wherein X₂ isselected from a bond and C₁₋₄alkylene; and each R₉ is independentlyselected from hydrogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₃₋₁₂cycloalkyl,C₃₋₈heterocycloalkyl and —NR₁₂R₁₃; wherein said cycloalkyl orheterocycloalkyl of R₉ is optionally substituted with 1 to 3 radicalsindependently selected from C₁₋₆alkyl, C₁₋₆alkoxy,halo-substituted-C₁₋₆alkoxy, halo-substituted-C₁₋₆alkyl and —NR₁₂R₁₃;wherein R₁₂ and R₁₃ are independently selected from hydrogen andC₁₋₆alkyl; or R₄ and R₅ together with the carbon atoms to which R₄ andR₅ are attached form C₃₋₈heteroaryl;

with the proviso that at least one of R₃, R₄, R₅, R₆ or R₇ has a sulfurdirectly linked to the phenyl ring of Formula I to which R₃, R₄, R₅, R₆and R₇ are attached; and the N-oxide derivatives, prodrug derivatives,protected derivatives, individual isomers and mixture of isomersthereof; and the pharmaceutically acceptable salts and solvates (e.g.hydrates) of such compounds.

In a second aspect, the present invention provides a pharmaceuticalcomposition which contains a compound of Formula I or a N-oxidederivative, individual isomers and mixture of isomers thereof; or apharmaceutically acceptable salt thereof, in admixture with one or moresuitable excipients.

In a third aspect, the present invention provides a method of treating adisease in an animal in which inhibition of kinase activity,particularly c-kit, PDGFRα and/or PDGFRβ activity, can prevent, inhibitor ameliorate the pathology and/or symptomology of the diseases, whichmethod comprises administering to the animal a therapeutically effectiveamount of a compound of Formula I or a N-oxide derivative, individualisomers and mixture of isomers thereof, or a pharmaceutically acceptablesalt thereof.

In a fourth aspect, the present invention provides the use of a compoundof Formula I in the manufacture of a medicament for treating a diseasein an animal in which kinase activity, particularly c-kit, PDGFRα and/orPDGFRβ activity, contributes to the pathology and/or symptomology of thedisease.

In a fifth aspect, the present invention provides a process forpreparing compounds of Formula I and the N-oxide derivatives, prodrugderivatives, protected derivatives, individual isomers and mixture ofisomers thereof, and the pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” as a group and as a structural element of other groups, forexample halo-substituted-alkyl and alkoxy, can be eitherstraight-chained or branched. C₁₋₄-alkoxy includes, methoxy, ethoxy, andthe like. Halo-substituted alkyl includes trifluoromethyl,pentafluoroethyl, and the like.

“Aryl” means a monocyclic or fused bicyclic aromatic ring assemblycontaining six to ten ring carbon atoms. For example, C₅₋₁₀aryl as usedin this application, includes but is not limited to phenyl or naphthyl,preferably phenyl. “Arylene” means a divalent radical derived from anaryl group.

“Heteroaryl” is a 5 to 15 member, unsaturated ring system containing 1to 3 heteroatoms independently selected from —O—, —N═, —NR—, —C(O)—,—S—, —S(O)— or —S(O)₂—, wherein R is hydrogen, C₁₋₄alkyl or a nitrogenprotecting group. For example, C₁₋₁₀heteroaryl (“C₁₋₁₀” meaning betweenone and ten carbon atoms are present in the ring system), as used inthis application includes, but is not limited to, pyrazolyl, pyridinyl,indolyl, thiazolyl, 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl,furanyl, benzo[b]furanyl, pyrrolyl, 1H-indazolyl,imidazo[1,2-a]pyridin-3-yl, oxazolyl, benzo[d]thiazol-6-yl,1H-benzo[d][1,2,3]triazol-5-yl, quinolinyl, 1H-indolyl,3,4-dihydro-2H-pyrano[2,3-b]pyridinyl and2,3-dihydrofuro[2,3-b]pyridinyl,3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl, etc.

“Cycloalkyl” means a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing the numberof ring atoms indicated. For example, C₃₋₁₀cycloalkyl includescyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

“Heterocycloalkyl” means a 3 to 8 member, saturated or partiallyunsaturated ring system containing 1 to 3 heteroatoms independentlyselected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)₂—, wherein Ris hydrogen, C₁₋₄alkyl or a nitrogen protecting group. For example,C₃₋₈heterocycloalkyl as used in this application to describe compoundsof the invention includes, but is not limited to, morpholino,pyrrolidinyl, azepanyl, piperidinyl, isoquinolinyl, tetrahydrofuranyl,pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinylone,1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.

“Halogen” (or halo) preferably represents chloro or fluoro, but may alsobe bromo or iodo.

“Kinase Panel” is a list of kinases comprising Abl(human), Abl(T315I),JAK2, JAK3, ALK, JNK1α1, ALK4, KDR, Aurora-A, Lck, Blk, MAPK1, Bmx,MAPKAP-K2, BRK, MEK1, CaMKII(rat), Met, CDK1/cyclinB, p70S6K, CHK2,PAK2, CK1, PDGFRα, CK2, PDK1, c-kit, Pim-2, c-RAF, PKA(h), CSK, PKBα,cSrc, PKCα, DYRK2, Plk3, EGFR, ROCK-I, Fes, Ron, FGFR3, Ros, Flt3,SAPK2α, Fms, SGK, Fyn, SIK, GSK3β, Syk, IGF-1R, Tie-2, IKKβ, TrKB, IR,WNK3, IRAK4, ZAP-70, ITK, AMPK(rat), LIMK1, Rsk2, Axl, LKB1, SAPK2β,BrSK2, Lyn (h), SAPK3, BTK, MAPKAP-K3, SAPK4, CaMKIV, MARK1, Snk,CDK2/cyclinA, MINK, SRPK1, CDK3/cyclinE, MKK4(m), TAK1, CDK5/p25,MKK6(h), TBK1, CDK6/cyclinD3, MLCK, TrkA, CDK7/cyclinH/MAT1, MRCKβ,TSSK1, CHK1, MSK1, Yes, CK1d, MST2, ZIPK, c-Kit (D816V), MuSK, DAPK2,NEK2, DDR2, NEK6, DMPK, PAK-4, DRAK1, PAR-1Bα, EphA1, PDGFRβ, EphA2,Pim-1, EphA5, PKBβ, EphB2, PKCβI, EphB4, PKCδ, FGFR1, PKCη, FGFR2, PKCθ,FGFR4, PKD2, Fgr, PKG1β, Flt1, PRK2, Hck, PYK2, HIPK2, Ret, IKKα, RIPK2,IRR, ROCK-II(human), JNK2α2, Rse, JNK3, Rsk1(h), PI3 Kγ, PI3 Kδ andPI3-Kβ. Compounds of the invention are screened against the kinase panel(wild type and/or mutation thereof) and inhibit the activity of at leastone of said panel members.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The c-kit gene encodes a receptor tyrosine kinase and the ligand for thec-kit receptor is called the stem cell factor (SCF), which is theprincipal growth factor for mast cell survival. The activity of thec-kit receptor protein tyrosine kinase is regulated in normal cells, andthe normal functional activity of the c-kit gene product is essentialfor maintenance of normal hematopoeisis, melanogenesis, genetogenesis,and growth and differentiation of mast cells. Mutations that causeconstitutive activation of c-kit kinase activity in the absence of SCFbinding are implicated in various diseases ranging from mastocyclosis tomalignant human cancers.

In one embodiment, with reference to compounds of Formula I, L isselected from —NC(O)— and —C(O)N—; R₁, R_(2a) and R_(2b) areindependently selected from hydrogen, hydroxy, C₃₋₈heterocycloalkyl,C₁₋₄alkyl, C₁₋₄alkoxy, halo-substituted-C₁₋₄alkoxy,halo-substituted-C₁₋₄alkyl, —NR₁₀R₁₁, —OX₁R₈; wherein X₁ is selectedfrom a bond and C₁₋₄alkylene; R₈ is C₃₋₁₂cycloalkyl; or R₁ and R₂together with the carbon atoms to which R₁ and R_(2a) are attached formphenyl; R₁₀ and R₁₁ are independently selected from hydrogen, C₁₋₄alkyl,C₁₋₄alkoxy, halo-substituted-C₁₋₄alkoxy, halo-substituted-C₁₋₄alkyl,C₃₋₈heterocycloalkyl, C₁₋₁₀heteroaryl; or R₁₀ and R_(1l) together withthe nitrogen to which R₁₀ and R₁₁ are both attached formC₃₋₈heterocycloalkyl or C₁₋₁₀heteroaryl; R₃ and R₇ are independentlyselected from hydrogen and halo; R₄ and R₆ are independently selectedfrom hydrogen, C₁₋₆alkyl, C₁₋₆alkoxy, halo-substituted-C₁₋₆alkoxy,—S(O)₀₋₂R₉; and R₅ is selected from halo, C₁₋₆alkyl, C₁₋₆alkoxy,halo-substituted-C₁₋₆alkoxy, C₃₋₈heterocycloalkyl, —OX₂R₉ and—S(O)₀₋₂R₉; wherein X₂ is selected from a bond and C₁₋₄alkylene; andeach R₉ is independently selected from hydrogen, C₁₋₆alkyl,C₃₋₁₂cycloalkyl and C₃₋₈heterocycloalkyl; wherein said cycloalkyl orheterocycloalkyl of R₉ is optionally substituted with 1 to 3 radicalsindependently selected from C₁₋₆alkyl and halo-substituted-C₁₋₆alkyl; orR₄ and R₅ together with the carbon atoms to which R₄ and R₅ are attachedform C₃₋₈heteroaryl, with the proviso that at least one of R₃, R₄, R₅,R₆ or R₇ has a sulfur directly linked to the phenyl ring.

In another embodiment, R₁ is selected from hydrogen, hydroxy,pyrrolidinyl, morpholino, methoxy, difluoro-methoxy, 2-fluoro-ethoxy andmethyl; or R₁ and R_(2a) together with the carbon atoms to which R₁ andR_(2a) are attached form phenyl (that is, the pyridyl ring of theMarkush structure is fused to a phenyl ring formed from R₁ and R_(2a)thereby creating an isoquinolinyl ring group).

In another embodiment, R₄ and R₆ are independently selected fromhydrogen, methyl-sulfonyl, methyl, 2-fluoro-ethoxy,methyl-piperazinyl-sulfonyl, propoxy, isobutoxy, 2,2,2-trifluoroethoxy,2,3-difluoro-2-(fluoromethyl)propoxy, butoxy and cyclopropyl-methoxy; R₅is selected from hydrogen, halo, methyl-sulfonyl, methyl, methoxy,ethoxy and morpholino; or R₄ and R₅ together with the carbon atoms towhich R₄ and R₅ are attached form thiazolyl.

In another embodiment are compounds selected from:N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;4-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzo[d]thiazole-6-carboxamide;2-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;3-methyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;4-methyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;4-ethoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(5-morpholinopyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(5-(pyrrolidin-1-yl)pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)-4-morpholinobenzamide;N-(3-(4-(5-methoxypyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;N-(3-(4-(5-(2-fluoroethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;N-(3-(4-(5-(cyclopropylmethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(5-methylpyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;3-(2-fluoroethoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)-3-propoxybenzamide;3-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;3-butoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;3-(cyclopropylmethoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(3-(4-(isoquinolin-4-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3-(methylsulfonyl)benzamide;4-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;N-(3-(4-(5-(difluoromethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(4-methylpiperazin-1-ylsulfonyl)benzamide;N-(3-(4-(5-hydroxypyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;3-isobutoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)-3-(2,2,2-trifluoroethoxy)benzamide;and3-(2,3-difluoro-2-(fluoromethyl)propoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide.

In one embodiment, the invention provides methods for treating a diseaseor condition modulated by the c-kit and PDGFRα/β kinase receptors,comprising administering compounds of Formula I, or pharmaceuticallyacceptable salts or pharmaceutical compositions thereof.

Examples of c-kit and/or PDGFRα/β mediated disease or conditions whichmay be mediated using the compounds and compositions of the inventioninclude but are not limited to a neoplastic disorder, an allergydisorder, an inflammatory disorder, an autoimmune disorder, agraft-versus-host disease, a Plasmodium related disease, a mast cellassociated disease, a metabolic syndrome, a CNS related disorder, aneurodegenerative disorder, a pain condition, a substance abusedisorder, a prion disease, a cancer, a heart disease, a fibroticdisease, idiopathic arterial hypertension (IPAH), or primary pulmonaryhypertension (PPH).

Examples of a mast cell associated disease which may be treated usingcompounds and compositions of the invention include but are not limitedto acne and Propionibacterium acnes, Fibrodysplasia ossificansprogressiva (FOP), inflammation and tissue destruction induced byexposure to chemical or biological weapons (such as anthrax andsulfur-mustard), Cystic fibrosis; renal disease, inflammatory muscledisorders, HIV, type II diabetes, cerebral ischemia, mastocytosis, drugdependence and withdrawal symptoms, CNS disorders, preventing andminimizing hair loss, bacterial infections, interstitial cystitis,inflammatory bowel syndrome (IBS), inflammatory bowel diseases (IBD),tumor angiogenesis, autoimmune diseases, inflammatory diseases, MultipleSclerosis (MS), allergic disorders (including asthma), and bone loss.

Examples of neoplastic disorders which may be treated using thecompounds and compositions of the invention include but are not limitedto mastocytosis, gastrointestinal stromal tumor, small cell lung cancer,non-small cell lung cancer, acute myelocytic leukemia, acute lymphocyticleukemia, myelodyplastic syndrome, chronic myelogenous leukemia,colorectal carcinoma, gastric carcinoma, testicular cancer, glioblastomaor astrocytoma.

Examples of allergy disorders which may be treated using the compoundsand compositions of the invention include but are not limited to asthma,allergic rhinitis, allergic sinusitis, anaphylactic syndrome, urticaria,angioedema, atopic dermatitis, allergic contact dermatitis, erythemanodosum, erythema multiforme, cutaneous necrotizing venulitis, insectbite skin inflammation, or blood sucking parasite infestation.

Examples of inflammatory disorders which may be treated using thecompounds and compositions of the invention include but are not limitedto rheumatoid arthritis, conjunctivitis, rheumatoid spondylitis,osteoarthritis or gouty arthritis.

Examples of autoimmune disorders which may be treated using thecompounds and compositions of the invention include but are not limitedto multiple sclerosis, psoriasis, intestine inflammatory disease,ulcerative colitis, Crohn's disease, rheumatoid arthritis,polyarthritis, local or systemic scleroderma, systemic lupuserythematosus, discoid lupus erythematosis, cutaneous lupus,dermatomyositis, polymyositis, Sjogren's syndrome, nodular panarteritis,autoimmune enteropathy or proliferative glomerulonephritis.

Examples of graft-versus-host diseases which may be treated using thecompounds and compositions of the invention include but are not limitedto organ transplantation graft rejection, such as kidneytransplantation, pancreas transplantation, liver transplantation, hearttransplantation, lung transplantation, or bone marrow transplantation.

Examples of metabolic syndrome which may be treated using the compoundsand compositions of the invention include but are not limited to type Idiabetes, type II diabetes, or obesity.

Examples of CNS related disorders which may be treated using thecompounds and compositions of the invention include but are not limitedto depression, dysthymic disorder, cyclothymic disorder, anorexia,bulimia, premenstrual syndrome, post-menopause syndrome, mental slowing,loss of concentration, pessimistic worry, agitation, self-deprecationand decreased libido, an anxiety disorder, a psychiatric disorder orschizophrenia.

Examples of depression conditions which may be treated using thecompounds and compositions of the invention include but are not limitedto bipolar depression, severe or melancholic depression, atypicaldepression, refractory depression, or seasonal depression. Examples ofanxiety disorders which may be treated using the compounds andcompositions of the invention include but are not limited to anxietyassociated with hyperventilation and cardiac arrhythmias, phobicdisorders, obsessive-compulsive disorder, posttraumatic stress disorder,acute stress disorder, and generalized anxiety disorder. Examples ofpsychiatric disorders which may be treated using the compounds andcompositions of the invention include but are not limited to panicattacks, including psychosis, delusional disorders, conversiondisorders, phobias, mania, delirium, dissociative episodes includingdissociative amnesia, dissociative fugue and dissociative suicidalbehavior, self-neglect, violent or aggressive behavior, trauma,borderline personality, and acute psychosis such as schizophrenia,including paranoid schizophrenia, disorganized schizophrenia, catatonicschizophrenia, and undifferentiated schizophrenia.

Examples of neurodegenerative disorder which may be treated using thecompounds and compositions of the invention include but are not limitedto Alzheimer's disease, Parkinson's disease, Huntington's disease, theprion diseases, Motor Neuron Disease (MND), or Amyotrophic LateralSclerosis (ALS).

Examples of pain conditions which may be treated using the compounds andcompositions of the invention include but are not limited to acute pain,postoperative pain, chronic pain, nociceptive pain, cancer pain,neuropathic pain or psychogenic pain syndrome.

Examples of substance use disorders which may be treated using thecompounds and compositions of the invention include but are not limitedto drug addiction, drug abuse, drug habituation, drug dependence,withdrawal syndrome or overdose.

Examples of cancers which may be treated using the compounds andcompositions of the invention include but are not limited to melanoma,colon cancer, gastrointestinal stromal tumor (GIST), small cell lungcancer, or other solid tumors.

Examples of fibrotic diseases which may be treated using the compoundsand compositions of the invention include but are not limited tohepatitis C(HCV), liver fibrosis, nonalcoholic steatohepatitis (NASH),scleroderma, cirrhosis in liver, pulmonary fibrosis, or bone marrowfibrosis.

In another embodiment, the invention provides methods for treating adisease or condition modulated by the c-kit and/or PDGFRα/β kinasereceptor, comprising administering compounds of Formula I, orpharmaceutically acceptable salts or pharmaceutical compositions thereof

Pharmacology and Utility

Compounds of the invention modulate the activity of kinases and, assuch, are useful for treating diseases or disorders in which kinases,contribute to the pathology and/or symptomology of the disease. Examplesof kinases that are inhibited by the compounds and compositionsdescribed herein and against which the methods described herein areuseful include, but are not limited to c-kit, PDGFRα, PDGFRβ, Lyn,MAPK14 (p38delta), PDGFRα, PDGFRβ, ARG, BCR-Abl, BRK, EphB, Fms, Fyn,KDR, LCK, b-Raf, c-Raf, SAPK2, Src, Tie2 and TrkB kinase.

Mast cells (MC) are tissue elements derived from a particular subset ofhematopoietic stem cells that produce a large variety of mediators mostof which having strong pro-inflammatory activities. Since MCs aredistributed in almost all the body sites, hypersecretion of mediators byactivated elements can lead to multiple organ failures. Mast cells are,therefore, central players involved in many diseases. The presentinvention relates to a method for treating mast cell associated diseasescomprising administering a compound capable of depleting mast cells or acompound inhibiting mast cell degranulation, to a human in need of suchtreatment. Such compounds can be chosen from c-kit inhibitors and moreparticularly non-toxic, selective and potent c-kit inhibitors.Preferably, said inhibitors are unable to promote death of IL-3dependent cells cultured in presence of IL-3.

Mast cell associated diseases include, but are not limited to: acne andPropionibacterium acnes (acne encompasses all forms of chronicinflammation of the skin including those induced by Propionibacteriumacnes); an extremely rare and disabling genetic disorder of connectivetissue known as Fibrodysplasia ossificans progressiva (FOP); thedetrimental effects of inflammation and tissue destruction induced byexposure to chemical or biological weapons (such as anthrax,sulfur-mustard, etc.); Cystic fibrosis (a lung, digestive andreproductive systems genetic disease); renal disease such as Acutenephritic syndrome, glomerulonephritis, renal amyloidosis, renalinterstitial fibrosis (the final common pathway leading to end-stagerenal disease in various nephropathies); inflammatory muscle disordersincluding myositis and muscular dystrophy; HIV (for example, depletingHIV infected mast cells can be a new route for treating HIV infectionand related diseases); treating type II diabetes, obesity and relateddisorders (mast cells regulate a number of the processes that contributeto the development of atherosclerosis, including hyperglycemia,hypercholesterolemia, hypertension, endothelial dysfunction, insulinresistance, and vascular remodeling; cerebral ischemia; mastocytosis (avery heterogeneous group of disorders characterized by an abnormalaccumulation of mast cells in different tissues, mainly in the skin andthe bone marrow, but also in spleen, liver, lymph nodes, and thegastrointestinal tract); drug dependence and withdrawal symptoms(particularly drug addiction, drug abuse, drug habituation, drugdependence, withdrawal syndrome and overdose); CNS disorders(particularly depression, schizophrenia, anxiety, migraine, memory loss,pain and neurodegenerative diseases); promoting hair growth (includingpreventing and minimizing hair loss); bacterial infections (particularlyinfections caused by FimH expressing bacteria); interstitial cystitis (achronic inflammation of the bladder wall resulting in tissue damage,especially at the interstices between the cells in the lining of thebladder); Inflammatory bowel diseases (generally applied to fourdiseases of the bowel, namely Crohn's disease, ulcerative colitis,indeterminate colitis, and infectious colitis); tumor angiogenesis;autoimmune diseases (particularly multiple sclerosis, ulcerativecolitis, Crohn's disease, rheumatoid arthritis and polyarthritis,scleroderma, lupus erythematosus, dermatomyositis, pemphigus,polymyositis, vasculitis and graft-versus host diseases); inflammatorydiseases such as rheumatoid arthritis (RA); Multiple Sclerosis (MS);allergic disorders (particularly asthma, allergic rhinitis, allergicsinusitis, anaphylactic syndrome, urticaria, angioedema, atopicdermatitis, allergic contact dermatitis, erythema nodosum, erythemamultiforme, cutaneous necrotizing venulitis and insect bite skininflammation, bronchial asthma); nasal polyposis; inflammatory boweldisease (IBD) and inflammatory bowel syndrome (IBS); and bone loss.

PDGF (Platelet-derived Growth Factor) is a very commonly occurringgrowth factor, which plays an important role both in normal growth andalso in pathological cell proliferation, such as is seen incarcinogenesis and in diseases of the smooth-muscle cells of bloodvessels, for example in atherosclerosis and thrombosis. Compounds of theinvention can inhibit PDGF receptor (PDGFR) activity and are, therefore,suitable for the treatment of: tumor diseases, such as gliomas,sarcomas, prostate tumors, and tumors of the colon, breast, and ovary;hypereosinophilia; fibrosis such as lung fibrosis, liver fibrosis andscleroderma; pulmonary hypertension; and cardiovascular diseases.

The Ras-Raf-MEK-ERK signaling pathway mediates cellular response togrowth signals. Ras is mutated to an oncogenic form in ˜15% of humancancer. The Raf family belongs to the serine/threonine protein kinaseand it includes three members, A-Raf, B-Raf and c-Raf (or Raf-1). Thefocus on Raf being a drug target has centered on the relationship of Rafas a downstream effector of Ras. However, recent data suggests thatB-Raf may have a prominent role in the formation of certain tumors withno requirement for an activated Ras allele (Nature 417, 949-954 (1 Jul.2002). In particular, B-Raf mutations have been detected in a largepercentage of malignant melanomas.

Existing medical treatments for melanoma are limited in theireffectiveness, especially for late stage melanomas. The compounds of thepresent invention also inhibit cellular processes involving b-Rafkinase, providing a new therapeutic opportunity for treatment of humancancers, especially for melanoma.

The compounds of the present invention also inhibit cellular processesinvolving c-Raf kinase. c-Raf is activated by the ras oncogene, which ismutated in a wide number of human cancers. Therefore inhibition of thekinase activity of c-Raf may provide a way to prevent ras mediated tumorgrowth [Campbell, S. L., Oncogene, 17, 1395 (1998)].

Compounds of the present invention, can be used to treat non-malignantproliferative disorders, such as atherosclerosis, thrombosis, psoriasis,scleroderma and fibrosis, as well as for the protection of stem cells,for example to combat the hemotoxic effect of chemotherapeutic agents,such as 5-fluoruracil, and in asthma.

Compounds of the present invention show useful effects in the treatmentof disorders arising as a result of transplantation, for example,allogenic transplantation, especially tissue rejection, such asespecially obliterative bronchiolitis (OB), i.e. a chronic rejection ofallogenic lung transplants. In contrast to patients without OB, thosewith OB often show an elevated PDGF concentration in bronchoalveolarlavage fluids.

Compounds of the present invention are also effective in diseasesassociated with vascular smooth-muscle cell migration and proliferation(where PDGF and PDGF-R often also play a role), such as restenosis andatherosclerosis. These effects and the consequences thereof for theproliferation or migration of vascular smooth-muscle cells in vitro andin vivo can be demonstrated by administration of the compounds of thepresent invention, and also by investigating its effect on thethickening of the vascular intima following mechanical injury in vivo.

The trk family of neurotrophin receptors (trkA, trkB, trkC) promotes thesurvival, growth and differentiation of the neuronal and non-neuronaltissues. The TrkB protein is expressed in neuroendocrine-type cells inthe small intestine and colon, in the alpha cells of the pancreas, inthe monocytes and macrophages of the lymph nodes and of the spleen, andin the granular layers of the epidermis (Shibayama and Koizumi, 1996).Expression of the TrkB protein has been associated with an unfavorableprogression of Wilms tumors and of neuroblastomas. TkrB is, moreover,expressed in cancerous prostate cells but not in normal cells. Thesignaling pathway downstream of the trk receptors involves the cascadeof MAPK activation through the Shc, activated Ras, ERK-1 and ERK-2genes, and the PLC-gamrnal transduction pathway (Sugimoto et al., 2001).

The kinase, c-Src transmits oncogenic signals of many receptors. Forexample, over-expression of EGFR or HER2/neu in tumors leads to theconstitutive activation of c-src, which is characteristic for themalignant cell but absent from the normal cell. On the other hand, micedeficient in the expression of c-src exhibit an osteopetrotic phenotype,indicating a key participation of c-src in osteoclast function and apossible involvement in related disorders.

The Tec family kinase, Bmx, a non-receptor protein-tyrosine kinase,controls the proliferation of mammary epithelial cancer cells.

Fibroblast growth factor receptor 3 was shown to exert a negativeregulatory effect on bone growth and an inhibition of chondrocyteproliferation. Thanatophoric dysplasia is caused by different mutationsin fibroblast growth factor receptor 3, and one mutation, TDII FGFR3,has a constitutive tyrosine kinase activity which activates thetranscription factor Stat1, leading to expression of a cell-cycleinhibitor, growth arrest and abnormal bone development (Su et al.,Nature, 1997, 386, 288-292). FGFR3 is also often expressed in multiplemyeloma-type cancers. Inhibitors of FGFR3 activity are useful in thetreatment of T-cell mediated inflammatory or autoimmune diseasesincluding but not limited to rheumatoid arthritis (RA), collagen IIarthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE),psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease,sarcoidosis, autoimmune uveitis, inflammatory bowel disease (Crohn's andulcerative colitis), celiac disease and myasthenia gravis.

The activity of serum and glucocorticoid-regulated kinase (SGK), iscorrelated to perturbed ion-channel activities, in particular, those ofsodium and/or potassium channels and compounds of the invention can beuseful for treating hypertension.

Lin et al (1997) J. Clin. Invest. 100, 8: 2072-2078 and P. Lin (1998)PNAS 95, 8829-8834, have shown an inhibition of tumor growth andvascularization and also a decrease in lung metastases during adenoviralinfections or during injections of the extracellular domain of Tie-2(Tek) in breast tumor and melanoma xenograft models. Tie2 inhibitors canbe used in situations where neovascularization takes placeinappropriately (i.e. in diabetic retinopathy, chronic inflammation,psoriasis, Kaposi's sarcoma, chronic neovascularization due to maculardegeneration, rheumatoid arthritis, infantile haemangioma and cancers).

Lck plays a role in T-cell signaling. Mice that lack the Lck gene have apoor ability to develop thymocytes. The function of Lck as a positiveactivator of T-cell signaling suggests that Lck inhibitors may be usefulfor treating autoimmune disease such as rheumatoid arthritis.

JNKs, along with other MAPKs, have been implicated in having a role inmediating cellular response to cancer, thrombin-induced plateletaggregation, immunodeficiency disorders, autoimmune diseases, celldeath, allergies, osteoporosis and heart disease. The therapeutictargets related to activation of the JNK pathway include chronicmyelogenous leukemia (CML), rheumatoid arthritis, asthma,osteoarthritis, ischemia, cancer and neurodegenerative diseases. As aresult of the importance of JNK activation associated with liver diseaseor episodes of hepatic ischemia, compounds of the invention may also beuseful to treat various hepatic disorders. A role for JNK incardiovascular disease such as myocardial infarction or congestive heartfailure has also been reported as it has been shown JNK mediateshypertrophic responses to various forms of cardiac stress. It has beendemonstrated that the JNK cascade also plays a role in T-cellactivation, including activation of the IL-2 promoter. Thus, inhibitorsof JNK may have therapeutic value in altering pathologic immuneresponses. A role for JNK activation in various cancers has also beenestablished, suggesting the potential use of JNK inhibitors in cancer.For example, constitutively activated JNK is associated with HTLV-1mediated tumorigenesis [Oncogene 13:135-42 (1996)]. JNK may play a rolein Kaposi's sarcoma (KS). Other proliferative effects of other cytokinesimplicated in KS proliferation, such as vascular endothelial growthfactor (VEGF), IL-6 and TNFα, may also be mediated by JNK. In addition,regulation of the c-jun gene in p210 BCR-ABL transformed cellscorresponds with activity of JNK, suggesting a role for JNK inhibitorsin the treatment for chronic myelogenous leukemia (CML) [Blood92:2450-60 (1998)].

Certain abnormal proliferative conditions are believed to be associatedwith raf expression and are, therefore, believed to be responsive toinhibition of raf expression. Abnormally high levels of expression ofthe raf protein are also implicated in transformation and abnormal cellproliferation. These abnormal proliferative conditions are also believedto be responsive to inhibition of raf expression. For example,expression of the c-raf protein is believed to play a role in abnormalcell proliferation since it has been reported that 60% of all lungcarcinoma cell lines express unusually high levels of c-raf mRNA andprotein. Further examples of abnormal proliferative conditions arehyper-proliferative disorders such as cancers, tumors, hyperplasia,pulmonary fibrosis, angiogenesis, psoriasis, atherosclerosis and smoothmuscle cell proliferation in the blood vessels, such as stenosis orrestenosis following angioplasty. The cellular signaling pathway ofwhich raf is a part has also been implicated in inflammatory disorderscharacterized by T-cell proliferation (T-cell activation and growth),such as tissue graft rejection, endotoxin shock, and glomerularnephritis, for example.

The stress activated protein kinases (SAPKs) are a family of proteinkinases that represent the penultimate step in signal transductionpathways that result in activation of the c-jun transcription factor andexpression of genes regulated by c-jun. In particular, c-jun is involvedin the transcription of genes that encode proteins involved in therepair of DNA that is damaged due to genotoxic insults. Therefore,agents that inhibit SAPK activity in a cell prevent DNA repair andsensitize the cell to agents that induce DNA damage or inhibit DNAsynthesis and induce apoptosis of a cell or that inhibit cellproliferation.

Mitogen-activated protein kinases (MAPKs) are members of conservedsignal transduction pathways that activate transcription factors,translation factors and other target molecules in response to a varietyof extracellular signals. MAPKs are activated by phosphorylation at adual phosphorylation motif having the sequence Thr-X-Tyr bymitogen-activated protein kinase kinases (MKKs). In higher eukaryotes,the physiological role of MAPK signaling has been correlated withcellular events such as proliferation, oncogenesis, development anddifferentiation. Accordingly, the ability to regulate signaltransduction via these pathways (particularly via MKK4 and MKK6) couldlead to the development of treatments and preventive therapies for humandiseases associated with MAPK signaling, such as inflammatory diseases,autoimmune diseases and cancer.

The family of human ribosomal S6 protein kinases consists of at least 8members (RSK1, RSK2, RSK3, RSK4, MSK1, MSK2, p70S6K and p70S6 Kb).Ribosomal protein S6 protein kinases play important pleotropicfunctions, among them is a key role in the regulation of mRNAtranslation during protein biosynthesis (Eur. J. Biochem 2000 November;267 (21): 6321-30, Exp Cell Res. Nov. 25, 1999; 253 (1):100-9, Mol CellEndocrinol. May 25, 1999; 151 (1-2):65-77). The phosphorylation of theS6 ribosomal protein by p70S6 has also been implicated in the regulationof cell motility (Immunol. Cell Biol. 2000 August; 78 (4):447-51) andcell growth (Prog. Nucleic Acid Res. Mol. Biol., 2000; 65:101-27), andhence, may be important in tumor metastasis, the immune response andtissue repair as well as other disease conditions.

The SAPK's (also called “jun N-terminal kinases” or “JNK's”) are afamily of protein kinases that represent the penultimate step in signaltransduction pathways that result in activation of the c-juntranscription factor and expression of genes regulated by c-jun. Inparticular, c-jun is involved in the transcription of genes that encodeproteins involved in the repair of DNA that is damaged due to genotoxicinsults. Agents that inhibit SAPK activity in a cell prevent DNA repairand sensitize the cell to those cancer therapeutic modalities that actby inducing DNA damage.

BTK plays a role in autoimmune and/or inflammatory disease such assystemic lupus erythematosus (SLE), rheumatoid arthritis, multiplevasculitides, idiopathic thrombocytopenic purpura (ITP), myastheniagravis, and asthma. Because of BTK's role in B-cell activation,inhibitors of BTK are useful as inhibitors of B-cell mediated pathogenicactivity, such as autoantibody production, and are useful for thetreatment of B-cell lymphoma and leukemia.

CHK2 is a member of the checkpoint kinase family of serine/threonineprotein kinases and is involved in a mechanism used for surveillance ofDNA damage, such as damage caused by environmental mutagens andendogenous reactive oxygen species. As a result, it is implicated as atumor suppressor and target for cancer therapy.

CSK influences the metastatic potential of cancer cells, particularlycolon cancer.

Fes is a non-receptor protein tyrosine kinase that has been implicatedin a variety of cytokine signal transduction pathways, as well asdifferentiation of myeloid cells. Fes is also a key component of thegranulocyte differentiation machinery.

Flt3 receptor tyrosine kinase activity is implicated in leukemias andmyelodysplastic syndrome. In approximately 25% of AML the leukemia cellsexpress a constitutively active form of auto-phosphorylated (p) FLT3tyrosine kinase on the cell surface. The activity of p-FLT3 confersgrowth and survival advantage on the leukemic cells. Patients with acuteleukemia, whose leukemia cells express p-FLT3 kinase activity, have apoor overall clinical outcome. Inhibition of p-FLT3 kinase activityinduces apoptosis (programmed cell death) of the leukemic cells.

Inhibitors of IKKα and IKKβ (1 & 2) are therapeutics for diseases whichinclude rheumatoid arthritis, transplant rejection, inflammatory boweldisease, osteoarthritis, asthma, chronic obstructive pulmonary disease,atherosclerosis, psoriasis, multiple sclerosis, stroke, systemic lupuserythematosus, Alzheimer's disease, brain ischemia, traumatic braininjury, Parkinson's disease, amyotrophic lateral sclerosis, subarachnoidhemorrhage or other diseases or disorders associated with excessiveproduction of inflammatory mediators in the brain and central nervoussystem.

Met is associated with most types of the major human cancers andexpression is often correlated with poor prognosis and metastasis.Inhibitors of Met are therapeutics for diseases which include cancerssuch as lung cancer, NSCLC (non small cell lung cancer), bone cancer,pancreatic cancer, skin cancer, cancer of the head and neck, cutaneousor intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, colon cancer, breast cancer,gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina or carcinoma of the vulva), Hodgkin's Disease, cancer ofthe esophagus, cancer of the small intestine, cancer of the endocrinesystem (e.g., cancer of the thyroid, parathyroid or adrenal glands),sarcomas of soft tissues, cancer of the urethra, cancer of the penis,prostate cancer, chronic or acute leukemia, solid tumors of childhood,lymphocytic lymphomas, cancer of the bladder, cancer of the kidney orureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis),pediatric malignancy, neoplasms of the central nervous system (e.g.,primary CNS lymphoma, spinal axis tumors, brain stem glioma or pituitaryadenomas), cancers of the blood such as acute myeloid leukemia, chronicmyeloid leukemia, etc, Barrett's esophagus (pre-malignant syndrome)neoplastic cutaneous disease, psoriasis, mycoses fungoides and benignprostatic hypertrophy, diabetes related diseases such as diabeticretinopathy, retinal ischemia and retinal neovascularization, hepaticcirrhosis, cardiovascular disease such as atherosclerosis, immunologicaldisease such as autoimmune disease and renal disease. Preferably, thedisease is cancer such as acute myeloid leukemia and colorectal cancer.

The Nima-related kinase 2 (Nek2) is a cell cycle-regulated proteinkinase with maximal activity at the onset of mitosis that localizes tothe centrosome. Functional studies have implicated Nek2 in regulation ofcentrosome separation and spindle formation. Nek2 protein is elevated 2-to 5-fold in cell lines derived from a range of human tumors includingthose of cervical, ovarian, prostate, and particularly breast.

p70S6K-mediated diseases or conditions include, but are not limited to,proliferative disorders, such as cancer and tuberous sclerosis.

In accordance with the foregoing, the present invention further providesa method for preventing or treating any of the diseases or disordersdescribed above in a subject in need of such treatment, which methodcomprises administering to said subject a therapeutically effectiveamount (See, “Administration and Pharmaceutical Compositions”, infra) ofa compound of Formula I or a pharmaceutically acceptable salt thereof.For any of the above uses, the required dosage will vary depending onthe mode of administration, the particular condition to be treated andthe effect desired.

Administration and Pharmaceutical Compositions

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount may vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors. Ingeneral, satisfactory results are indicated to be obtained systemicallyat daily dosages of from about 0.03 to 2.5 mg/kg per body weight. Anindicated daily dosage in the larger mammal, e.g. humans, is in therange from about 0.5 mg to about 100 mg, conveniently administered, e.g.in divided doses up to four times a day or in retard form. Suitable unitdosage forms for oral administration comprise from ca. 1 to 50 mg activeingredient.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal, inhaledor suppository form. Pharmaceutical compositions comprising a compoundof the present invention in free form or in a pharmaceuticallyacceptable salt form in association with at least one pharmaceuticallyacceptable carrier or diluent can be manufactured in a conventionalmanner by mixing, granulating or coating methods. For example, oralcompositions can be tablets or gelatin capsules comprising the activeingredient together with a) diluents, e.g., lactose, dextrose, sucrose,mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g.,silica, talcum, stearic acid, its magnesium or calcium salt and/orpolyethyleneglycol; for tablets also c) binders, e.g., magnesiumaluminum silicate, starch paste, gelatin, tragacanth, methylcellulose,sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations may also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such maycontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

Compounds of the invention can be administered in therapeuticallyeffective amounts in combination with one or more therapeutic agents(pharmaceutical combinations). For example, synergistic effects canoccur with other asthma therapies, for example, steroids and leukotrieneantagonists.

For example, synergistic effects can occur with other immunomodulatoryor anti-inflammatory substances, for example when used in combinationwith cyclosporin, rapamycin, or ascomycin, or immunosuppressantanalogues thereof, for example cyclosporin A (CsA), cyclosporin G,FK-506, rapamycin, or comparable compounds, corticosteroids,cyclophosphamide, azathioprine, methotrexate, brequinar, leflunomide,mizoribine, mycophenolic acid, mycophenolate mofetil, broncho dilators,15-deoxyspergualin, immunosuppressant antibodies, especially monoclonalantibodies for leukocyte receptors, for example MHC, CD2, CD3, CD4, CD7,CD25, CD28, B7, CD45, CD58 or their ligands, or other immunomodulatorycompounds, such as CTLA41g. Where the compounds of the invention areadministered in conjunction with other therapies, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the condition beingtreated and so forth.

The invention also provides for a pharmaceutical combinations, e.g. akit, comprising a) a first agent which is a compound of the invention asdisclosed herein, in free form or in pharmaceutically acceptable saltform, and b) at least one co-agent. The kit can comprise instructionsfor its administration.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of Formula I and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of Formula I and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the 2compounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of 3 or more activeingredients.

Processes for Making Compounds of the Invention

The present invention also includes processes for the preparation ofcompounds of the invention. In the reactions described, it can benecessary to protect reactive functional groups, for example hydroxy,amino, imino, thio or carboxy groups, where these are desired in thefinal product, to avoid their unwanted participation in the reactions.Conventional protecting groups can be used in accordance with standardpractice, for example, see T. W. Greene and P. G. M. Wuts in “ProtectiveGroups in Organic Chemistry”, John Wiley and Sons, 1991.

Compounds of Formula I, wherein L is —NHC(O)—, can be prepared byproceeding as in the following Reaction Schemes I:

wherein R₁, R_(2a), R_(2b), R₃, R₄, R₅, R₆ and R₇ are as described inthe Summary of the Invention. A compound of Formula I can be prepared byreacting of a compound of formula 2 with a compound of formula 3 in thepresence of a suitable solvent (for example, DMF, and the like), asuitable coupling agent (for example, HATU, and the like) and a suitablebase (for example, DIEA, and the like). The reaction is carried out in atemperature range of about 0° C. to about 60° C. and can take up to 24hours to complete.

Compounds of Formula I, wherein L is —C(O)NH—, can be prepared byproceeding as in the following Reaction Schemes II:

wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are as described in the Summary ofthe Invention. A compound of Formula I can be prepared by reacting of acompound of formula 4 with a compound of formula 5 in the presence of asuitable solvent (for example, DMF, and the like), a suitable couplingagent (for example, HATU, and the like) and a suitable base (forexample, DIEA, and the like). The reaction is carried out in atemperature range of about 0° C. to about 60° C. and can take up to 24hours to complete.

Detailed examples of the synthesis of compounds of formula I can befound in the Examples, infra.

Additional Processes for Making Compounds of the Invention

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase. Alternatively, the salt forms of the compounds of the inventioncan be prepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Compounds of the invention in unoxidized form can be prepared fromN-oxides of compounds of the invention by treating with a reducing agent(e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride,sodium borohydride, phosphorus trichloride, tribromide, or the like) ina suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueousdioxane, or the like) at 0 to 80° C.

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., (1994), Bioorganic and Medicinal ChemistryLetters, Vol. 4, p. 1985). For example, appropriate prodrugs can beprepared by reacting a non-derivatized compound of the invention with asuitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3^(rd) edition, John Wiley and Sons, Inc.,1999.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxin, tetrahydrofuranor methanol.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomers. While resolution of enantiomers can be carried outusing covalent diastereomeric derivatives of the compounds of theinvention, dissociable complexes are preferred (e.g., crystallinediastereomeric salts). Diastereomers have distinct physical properties(e.g., melting points, boiling points, solubilities, reactivity, etc.)and can be readily separated by taking advantage of thesedissimilarities. The diastereomers can be separated by chromatography,or preferably, by separation/resolution techniques based upondifferences in solubility. The optically pure enantiomer is thenrecovered, along with the resolving agent, by any practical means thatwould not result in racemization. A more detailed description of thetechniques applicable to the resolution of stereoisomers of compoundsfrom their racemic mixture can be found in Jean Jacques, Andre Collet,Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John WileyAnd Sons, Inc., 1981.

In summary, the compounds of Formula I can be made by a process, whichinvolves:

(a) those of reaction schemes I and II, and

(b) optionally converting a compound of the invention into apharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention toa non-salt form;

(d) optionally converting an unoxidized form of a compound of theinvention into a pharmaceutically acceptable N-oxide;

(e) optionally converting an N-oxide form of a compound of the inventionto its unoxidized form;

(f) optionally resolving an individual isomer of a compound of theinvention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the inventioninto a pharmaceutically acceptable prodrug derivative; and

(h) optionally converting a prodrug derivative of a compound of theinvention to its non-derivatized form.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or can be prepared analogously tomethods known in the art or as disclosed in the Examples hereinafter.

One of skill in the art will appreciate that the above transformationsare only representative of methods for preparation of the compounds ofthe present invention, and that other well known methods can similarlybe used.

EXAMPLES

The present invention is further exemplified, but not limited, by thefollowing examples that illustrate the preparation of compounds ofFormula I according to the invention.

Experimental Preparation of Intermediates Synthesis of6-methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine 5

To 2-amino-4-nitro toluene 1 (0.033 mol) in n-butanol (29 mL) is addednitric acid (2.1 g, 65% in water) followed by cyanamide solution inwater (2 mL, 0.047 mmol). The resulting mixture is heated at reflux for25 h. After cooling to 0° C., filtration and washing with1:1=ethanol:diethyl ether (30 mL), 2-methyl-5-nitrophenyl guanidinenitrate 2 is obtained. To 2-methyl-5-nitrophenyl guanidine 2 (0.0074mol) in isopropanol (15 ME) is added 3 (0.0074 mol) and sodium hydroxideflakes (0.008 mol). The resulting mixture is heated at reflux for 12 h.After cooling to 0° C., the product is collected by filtration andwashed with isopropanol (6 mL) and methanol (3 mL) to afford 4. ¹H NMR(400 MHz, d₆-DMSO) δ 9.31 (s, 1H), 9.24 (s, 1H), 8.78 (m, 1H), 8.70 (m,1H), 8.61 (m, 1H), 8.47 (m, 1H), 7.88 (m, 1H), 7.55 (m, 3H), 2.39 (s,3H). MS (m/z) (M+1)⁺: 278.2.

Reactant 3 can be obtained by the following procedures. A mixture of3-acetylpyridine (2.47 mol) and N,N-dimethylformamide dimethylacetal(240 mL) is heated at reflux for 16 h. The solvent is removed in vacuoand hexanes (100 mL) is added to the residue to crystallize a solid. Thesolid is recrystallized from methylene chloride-hexanes to give3-dimethylamino-1-(3-pyridyl)-2-propen-1-one 3.

¹H NMR (400 MHz, d-chloroform) δ 9.08 (d, J=2.4 Hz, 1H), 8.66 (m, 1H),8.20 (m, 1H), 7.87 (m, 1H), 7.37 (m, 1H), 5.68 (d, J=16.4 Hz, 1H), 3.18(s, 3H), 2.97 (s, 3H).

A reactor is charged with concentrated hydrochloric acid (17 mL)followed by stannous chloride dehydrate (0.03 mol). The mixture isstirred for 10 min and then cooled to 0-5° C. A solution of compound 4(5.6 mmol) in ethyl acetate (3 mL) is then slowly added (during 3-4minutes) while maintaining the temperature at 0-5° C. The reactionmixture is brought to rt and stirred for 1.5 h. To this is added water(50 mL) followed by a slow addition of 50% sodium hydroxide solution (40mL). The resulting mixture is extracted with chloroform (2×25 mL). Theorganic layer is washed with water thoroughly and evaporated. Theresidue is dissolved in ethyl acetate (2 mL), cooled to 0-10° C. andmaintained at this temperature for 1 h. The resulting precipitate iscollected by filtration and washed with ethyl acetate (1 mL) to provide1.0 g of 5. ¹H NMR (400 MHz, d-chloroform) δ 9.26 (d, J=2.0 Hz, 1H),8.71 (m, 1H), 8.48 (d, J=6.8 Hz, 1H), 8.34 (m, 1H), 7.59 (d, J=4.0 Hz,1H), 7.41 (m, 1H), 7.12 (m, 1H), 7.04 (m, 1H), 6.42 (m, 1H), 3.50 (bs,2H), 2.24 (s, 3H).

Synthesis of4-(5-bromopyridin-3-yl)-N-(2-methyl-5-nitrophenyl)pyrimidin-2-amine 8

1-(5-Bromo-pyridin-3-yl)-3-dimethylamino-propenone 7 is prepared from1-(5-bromo-pyridin-3-yl)-ethanone with a similar protocol used for thesynthesis of 3. LC/MS (m/z) (M+1)⁺: 386.1, 388.1.

Condensation with N-(2-methyl-5-nitro-phenyl)-guanidine nitrate with aprotocol similar to the one used for the preparation of 4 affords[4-(5-bromo-pyridin-3-yl)-pyrimidin-2-yl]-(2-methyl-5-nitro-phenyl)-amine8. ¹H NMR (400 MHz, d-CDCl₃) δ 9.33 (d, J=2.4 Hz, 1H), 9.09 (d, J=1.6Hz, 1H), 8.74 (d, J=2.4 Hz, 1H), 8.61 (t, J=2.0 Hz, 1H), 8.53 (d, J=4.8Hz, 1H), 8.01 (s, 1H), 7.82 (dd, J=8.4, 2.4 Hz, 1H), 7.29 (d, J=8.0 Hz,1H), 7.22 (d, J=4.8 Hz, 1H), 7.10 (s, 1H), 2.41 (s, 3H). LC/MS (m/z)(M+1)⁺: 255.0, 257.0.

Synthesis of6-methyl-N1-(4-(5-(pyrrolidin-1-yl)pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine10

(2-Methyl-5-nitro-phenyl)-[4-(5-pyrrolidin-1-yl-pyridin-3-yl)-pyrimidin-2-yl]-amine9 is prepared by mixing 8 (0.40 g, 1.04 mmol), pyrrolidine (3.12 mmol),K₃PO₄ (0.44 g, 2.08 mmol), CuI (38.5 mg, 0.10 mmol) and L-proline aminoacid (48.1 mg, 0.21 mmol) in DMSO (8 ml) and heating at 160° C. in amicrowave oven for 20 min. The cooled mixture is partitioned betweenwater and ethyl acetate. The organic layer is separated, and the aqueouslayer is extracted with ethyl acetate. The combined organic layers arewashed with brine, dried over Na₂SO₄, and concentrated in vacuo. Theresidue is dissolved in DMSO (4 mL) and purified with HPLC to affordpure product 9. ¹H NMR (400 MHz, d-CDCl₃) δ 9.23 (d, J=2.4 Hz, 1H), 8.63(d, J=5.2 Hz, 1H), 8.58 (s, 1H), 8.10 (s, 1H), 8.04 (s, 1H), 7.90 (dd,J=8.4, 2.4 Hz, 1H), 7.44 (s, 1H), 7.39 (d, J=8.4 Hz, 1H), 7.30 (d, J=5.2Hz, 1H), 3.43-3.48 (m, 4H), 2.49 (s, 3H), 2.12-2.16 (m, 4H). LC/MS (m/z)(M+1)⁺: 377.2.

4-Methyl-N-3-[4-(5-pyrrolidin-1-yl-pyridin-3-yl)-pyrimidin-2-yl]-benzene-1,3-diamine10 is prepared by hydrogenation of 9 in EtOH in the presence of 10% Pd/Cand 1 atm hydrogen at rt (30%). Solvent is removed after filtrationthrough Celite and the product is used without further purification.LC/MS (m/z) (M+1)⁺: 347.2.

By substituting the pyrrolidine with other amines (e.g. morpholine)different intermediates similar to 9 and 10 can be synthesized.

Synthesis ofN1-(4-(5-methoxypyridin-3-yl)pyrimidin-2-yl)-6-methylbenzene-1,3-diamine

A mixture of 3-bromo-5-methoxypyridine (3.0 g, 16 mmol) and Pd(PPh₃)₄ isdegassed several times and purged with N₂. Tributyl(1-ethoxyvinyl)tin(7.04 mL, 20.8 mmol) and toluene (15 mL) are added to the mixture. Theresulting reaction mixture is heated at 150° C. for 30 minutes in amicrowave oven. After the reaction is complete, the reaction mixture isfiltered through a pad of Celite, washed with MeOH and concentrated togive a residue. To the above residue is added HCl (1N, 25 mL) and THF(25 mL) and the mixture is stirred at room temperature for 2 h. Thesolvent is removed in vacuo and the residue is dissolved in EtOAc. Theorganic layer is washed with Na₂CO₃ solution, dried with Na₂SO₄,filtered and concentrated to afford a residue which is purified bysilica gel column chromatography (EtOAc:hexanes=1:1) to afford 11 as alight yellow solid. ¹H NMR (400 MHz, d6-DMSO) δ 8.75 (d, J=1.6 Hz, 1H),8.52 (d, J=3.2 Hz, 1H), 7.74 (dd, J=1.6, 3.2 Hz, 1H), 3.91 (s, 3H), 2.64(s, 3H). MS (m/z) (M+1)⁺: 152.1.

3-(Dimethylamino)-1-(5-methoxypyridin-3-yl)prop-2-en-1-one 12 isprepared from 11 by reacting with diethoxy-N,N-dimethylmethanamine usinga similar procedure for the preparation of 3. ¹H NMR (400 MHz, d6-DMSO)δ 8.69 (d, J=2.0 Hz, 1H), 8.37 (d, J=2.8 Hz, 1H), 7.76 (d, J=12.0 Hz,1H), 7.70 (dd, J=1.6, 2.8 Hz, 1H), 5.86 (d, J=12.0 Hz, 1H), 3.89 (s,3H), 3.17 (s, 3H), 2.95 (s, 3H). MS (m/z) (M+1)⁺: 207.1

Condensation of 12 with N-(2-methyl-5-nitro-phenyl)-guanidine nitrate 2using a similar procedure for the preparation of 4 affords4-(5-methoxypyridin-3-yl)-N-(2-methyl-5-nitrophenyl)pyrimidin-2-amine13. ¹H NMR (400 MHz, d6-DMSO) δ 9.23 (s, 1H), 8.93 (d, J=1.2 Hz, 1H),8.80 (d, J=2.0 Hz, 1H), 8.63 (d, J=5.2 Hz, 1H), 8.44 (d, J=2.8 Hz, 1H),8.00 (s, 1H), 7.91 (dd, J=2.0, 8.4 Hz, 1H), 7.62 (d, J=5.2 Hz, 1H), 7.52(d, J=8.4 Hz, 1H), 3.90 (s, 3H), 2.44 (s, 3H). MS (m/z) (M+1)⁺: 338.1

N3-[4-(5-Methoxy-pyridin-3-yl)-pyrimidin-2-yl]-4-methyl-benzene-1,3-diamine14 is prepared by hydrogenation of 13 using a similar protocol for thepreparation of 10. ¹H NMR (400 MHz, d6-DMSO) δ 8.90 (d, J=1.6 Hz, 1H),8.71 (s, 1H), 8.51 (d, J=5.2 Hz, 1H), 8.45 (d, J=2.8 Hz, 1H), 8.01 (dd,J=2.0, 3.2 Hz, 1H), 7.43 (d, J=5.2 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 6.87(d, J=2.4 Hz, 1H), 6.38 (dd, J=2.4, 8.0 Hz, 1H), 4.87 (s, 2H), 3.95 (s,3H), 2.12 (s, 3H). MS (m/z) (M+1)⁺: 308.1

Synthesis ofN1-(4-(5-(2-fluoroethoxy)pyridin-3-yl)pyrimidin-2-yl)-6-methylbenzene-1,3-diamine17

Compound 13 (220 mg, 0.65 mmol) is dissolved in DCM and treated withBBr₃ (3.25 mmol) at −78° C. overnight. The mixture is diluted with DCMand washed with 1 N aqueous NaOH. The resultant aqueous phase isacidified with excess 1N HCl and extracted with DCM. Concentration gives15 which is used without further purification. LC/MS (m/z) (M+1)⁺:324.2.

Compound 15 (87.0 mg, 0.27 mmol) is heated with CsHCO₃ (103.6 mg, 0.54mmol) and 1-bromo-2-fluoro-ethane (102.0 mg, 0.80 mmol) in acetonitrile(2.0 mL) at reflux overnight. The mixture is purified by preparativeLC/MS to afford 16. LC/MS (m/z) (M+1)⁺: 370.1.

Compound 16 (50.0 mg, 0.14 mmol) is heated with SnCl₂.H₂O (77.2 mg, 0.41mmol) in EtOH (2.0 mL) at reflux for 1 h. The mixture is dissolved inNaOH (1N, 50 mL) and extracted with DCM. The organic layer is separated,dried over Na₂SO₄, filtered and concentrated to give a crude productwhich is used without further purification. LC/MS (m/z) (M+1)⁺: 340.1.

By substituting 1-bromo-2-fluoro-ethane with other alkyl halides (e.g.bromomethylcyclopropane) different intermediates similar to 16 and 17can be synthesized.

Synthesis ofN-(2-methyl-5-nitrophenyl)-4-(5-methylpyridin-3-yl)pyrimidin-2-amine 21

5-Methyl-nicotinonitrile (2.08 g, 17.6 mmol) is heated at reflux in dryTHF (20 mL) with MgMeBr (3 M solution in Et₂O, 10 mL, 30 mmol) for 2 h.After cooling down, aq. Na₂CO₃ is added slowly to quench the reaction.Extractions with DCM affords the crude mixture which is purified bysilica gel chromatography to yield 1-(5-methyl-pyridin-3-yl)-ethanone 18(0.7 g, 30%). LC/MS (m/z) (M+1)⁺: 136.1.

1-(5-Methyl-pyridin-3-yl)-ethanone 19 is prepared using a similarprocedure for the preparation of 3 by reacting 18 withdiethoxy-N,N-dimethylmethanamine. ¹H NMR (400 MHz, d6-DMSO) δ 8.87 (d,J=1.2 Hz, 1H), 8.49 (d, J=1.2 Hz, 1H), 8.02-8.04 (m, 1H), 7.75 (d,J=12.0 Hz, 1H), 5.85 (d, J=12.0 Hz, 1H), 3.16 (s, 3H), 2.94 (s, 3H),2.35 (s, 3H). LC/MS (m/z) (M+1)⁺: 191.2.

To 2-methyl-5-nitrophenyl guanidine 2 (2.0 mmol) in n-butanol (15 mL) isadded 19 (2.0 mmol) and sodium hydroxide flakes (2.0 mmol). Theresulting mixture is heated at 180° C. for 40 min in a microwave oven.After cooling to 0° C., the product is collected by filtration andwashed with ether (20 mL) and methanol (10 mL) to afford 20. ¹H NMR (400MHz, d6-DMSO) δ 9.19 (s, 1H), 9.13 (d, J=1.6 Hz, 1H), 8.91 (d, J=2.0 Hz,1H), 8.63 (d, J=5.2 Hz, 1H), 8.57 (d, J=1.2 Hz, 1H), 8.37 (s, 1H), 7.90(dd, J=8.0, 2.4 Hz, 1H), 7.59 (d, J=5.2 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H),2.45 (s, 3H), 2.41 (s, 3H). LC/MS (m/z) (M+1)⁺: 322.2.

Compound 21 is prepared by hydrogenation of 20 in MeOH in the presenceof 10% Pd/C and hydrogen balloon at rt. Solvent is removed afterfiltration through Celite and the product is used without furtherpurification (95%). LC/MS (m/z) (M+1)⁺: 292.1.

Synthesis of3-hydroxy-4-methanesulfonyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide23

6-Methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine 5 (578mg, 2.09 mmol), 4-iodo-3-hydroxy-benzoic acid (587 mg, 2.22 mmol) andHATU (960 mg, 2.52 mmol) are dissolved in dry DMF (5 mL) at rt.Diisopropylethylamine (0.732 mL, 4.2 mmol) is added dropwise to thesolution. After 30 min, the mixture is added slowly to saturated aqueousNaHCO₃. The solid is filtered, washed with water and dried under vacuumovernight to afford the product 22 as a tanish solid (630 mg, 60%).LC/MS (M+1): 524.1.

Compound 22 (580 mg, 1.11 mmol), CuI (42 mg, 0.272 mmol), L-proline (61mg, 0.444 mmol), 1 N NaOH (0.5 mL) and NaSO₂Me (267 mg, 2.22 mmol) aresuspended in DMSO (3 mL) and heated at 180° C. in a microwave oven for25 min. The mixture is diluted with concentrate aqueous ammonia andwashed by EtOAc. 1 N aq. HCl is added to aqueous phase until pH=7 andthe solid precipitate is collected by filtration to afford product 23(80%). LC/MS (M+1): 476.1.

Synthesis of Final Compounds Type AN-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3(methylsulfonyl)benzamide A1

6-Methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine 5 (5mmol), 3-(methylsulfonyl)benzoic acid (6 mmol) and HATU (6 mmol) aredissolved in dry DMF (5 mL) at rt. Diisopropylethylamine (6 mmol) isadded dropwise to the solution. After 30 min, the mixture is addedslowly to saturated aqueous NaHCO₃. The solid is filtered, washed withwater and dried under vacuum overnight to afford the product A1 as alight yellow solid. ¹H NMR (400 MHz, d₆-DMSO) δ 10.5 (s, 1H), 9.33 (s,1H), 9.04 (s, 1H), 8.75 (d, J=1.7 Hz, 1H), 8.63 (d, J=7.5 Hz, 1H), 8.55(d, J=5.0 Hz, 1H), 8.47 (s, 1H), 8.29 (d, J=8 Hz, 1H), 8.13 (d, J=8.6Hz, 1H), 8.11 (s, H), 7.83 (t, J=8 Hz, 1H), 7.66 (dd, J=7.3, 5.3 Hz,1H), 7.47 (m, 2H), 7.25 (d, J=8.4 Hz, 1H), 3.29 (s, 3H), 2.55 (s, 6H,2Me-HOAc), 2.24 (s, 3H). LC/MS (m/z) (M+1)⁺: 460.2.

Similar procedures are used to prepare compounds A2-A5 using LC/MS forpurification.

N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamideA2

¹H NMR (400 MHz, DMSO) δ 10.52 (s, 1H), 9.26 (s, 1H), 8.90 (s, 1H), 8.69(s, 1H), 8.61 (d, J=8.0 Hz, 1H), 8.50 (d, J=4.8 Hz, 1H), 8.08-8.14 (m,3H), 8.00-8.07 (m, 2H), 7.65 (m, 1H), 7.40 (m, 1H), 7.23 (m, 1H), 3.22(s, 3H), 2.21 (s, 3H). LC/MS (m/z) (M+1)⁺: 460.2.

4-Chloro-3-methanesulfonyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamideA3

¹H NMR (400 MHz, d₆-DMSO) δ 10.6 (s, 1H), 9.3 (s, 1H), 9.04 (s, 1H),8.72 (d, J=3.8 Hz, 1H), 8.55 (m, 3H), 8.3 (dd, J=8.3, 2.1 Hz, 1H), 8.08(d, J=1.5 Hz, 1H), 7.94 (d, J=8.3 Hz, 1H), 7.6 (dd, J=7.9, 4.8 Hz, 1H),7.48 (m, 2H), 7.24 (d, J=8.3 Hz, 1H), 3.44 (s, 3H), 2.24 (s, 3H). LC/MS(m/z) (M+1)⁺: 494.1.

N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)benzo[d]thiazole-6-carboxamideA4

¹H NMR (400 MHz, DMSO) δ 10.20 (s, 1H), 9.31 (s, 1H), 9.02 (s, 1H), 8.74(d, J=3.2 Hz, 1H), 8.58 (m, 1H), 8.53 (d, J=4.8 Hz, 1H), 8.06 (s, 1H),7.62 (m, 1H), 7.45 (d, J=5.2 Hz, 1H), 7.41 (d, J=7.2 Hz, 1H), 7.15-7.22(m, 3H), 7.09-7.13 (m, 1H), 2.22 (s, 3H). LC/MS (m/z) (M+1)⁺: 439.2.

N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-2-chloro-4-(methylsulfonyl)benzamideA5

¹H NMR (400 MHz, d₆-DMSO) δ 10.65 (s, 1H), 9.29 (s, 1H), 9.03 (s, 1H),8.72 (d, J=4.4 Hz, 1H), 8.55 (m, 2H), 8.06 (s, 1H), 8.00 (d, J=9.2, 1H),7.87 (d, J=8.0 Hz, 1H), 7.60 (m, 1H), 7.45 (d, J=4.8 Hz, 1H), 7.37 (d,J=8.0 Hz, 1H), 7.24 (d, J=8.4 Hz, 1H), 3.34 (s, 3H), 2.24 (s, 3H). LC/MS(m/z) (M+1)⁺: 494.1.

4-Methanesulfonyl-3-methyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamideA6

Intermediate 6 is prepared similar to A1. Compound 6 (0.1 mmol), CuI(0.075 mmol), L-proline (0.1 mmol), 1 N NaOH (0.1 mL) and NaSO₂Me (0.15mmol) are suspended in DMSO (0.5 mL) and heated at 180° C. in amicrowave oven for 5 min. The mixture is purified by preparative LC/MSto afford A6. ¹H NMR (400 MHz, DMSO) δ 10.45 (s, 1H), 9.24 (s, 1H), 8.89(s, 1H), 8.67 (d, J=2.0 Hz, 1H), 8.49 (m, 2H), 8.09 (s, 1H), 8.01 (d,J=8.4, 1H), 7.90-7.93 (m, 2H), 7.56 (m, 1H), 7.39 (d, J=5.6 Hz, 2H),7.23 (d, J=8.4 Hz, 1H), 3.32 (s, 3H), 2.70 (s, 3H), 2.21 (s, 3H). LC/MS(m/z) (M+1)⁺: 474.2.

Similar procedures are used to prepare A7, A8

N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-methyl-3-(methylsulfonyl)benzamideA7

¹H NMR (400 MHz, DMSO) δ 10.46 (s, 1H), 9.23 (s, 1H), 8.87 (d, J=7.2 Hz,1H), 8.67 (m, 1H), 8.50 (m, 2H), 8.05 (d, J=12.8 Hz, 1H), 7.58 (m, 2H),7.39 (m, 2H), 7.29 (m, 1H), 7.22 (m, 2H), 3.23 (s, 3H), 2.69 (s, 3H),2.21 (s, 3H). LC/MS (m/z) (M+1)⁺: 474.2.

N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-ethoxy-3-(methylsulfonyl)benzamideA8

¹H NMR (400 MHz, DMSO) δ 10.37 (s, 1H), 9.23 (s, 1H), 8.89 (s, 1H), 8.66(d, J=3.6 Hz, 1H), 8.48 (m, 2H), 8.37 (d, J=2.0 Hz, 1H), 8.25 (d, J=8.8Hz, 1H), 8.05 (s, 1H), 7.55 (m, 1H), 7.39 (m, 3H), 7.22 (d, J=8.0 Hz,1H), 4.30 (m, 2H), 3.27 (s, 3H), 2.20 (s, 3H), 1.41 (t, J=6.4 Hz, 3H).LC/MS (m/z) (M+1)⁺: 504.2.

Type B4-Methanesulfonyl-N-{4-methyl-3-[4-(5-pyrrolidin-1-yl-pyridin-3-yl)-pyrimidin-2-ylamino]-phenyl}-benzamideB1

Final compound B1 is prepared from 10 with a procedure similar to theone used for the preparation of A1. ¹H NMR (400 MHz, d₆-DMSO) δ 10.5 (s,1H), 9.12 (s, 1H), 8.6 (s, 1H), 8.59 (s, 1H), 8.18 (m, 3H), 8.09 (m,3H), 7.89 (s, 1H), 7.54 (d, J=5.1 Hz, 1H), 7.5 (dd, J=8.2, 2.1 Hz, 1H),7.23 (d, J=8.5 Hz, 1H), 3.3 (m, 4H), 3.29 (s, 3H), 2.24 (s, 3H), 1.86(m, 4H). LC/MS (m/z) (M+1)⁺: 530.1.

Type C3-Methanesulfonyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-morpholin-4-yl-benzamideC1

A3 (20 mg, 0.04 mmol) and morpholine (0.07 mL, 0.8 mmol) are heated at130° C. in a microwave oven for 30 min. Purification by preparativeLC/MS affords C1. LC/MS (m/z) (M+1)⁺: 545.3.

Type D4-Methanesulfonyl-N-{3-[4-(5-methoxy-pyridin-3-yl)-pyrimidin-2-ylamino]-4-methyl-phenyl}-benzamideD1

Final compound D1 is obtained from 14 using a similar procedure to thepreparation of A1. ¹H NMR (400 MHz, d₄-MeOH) δ 9.29 (s, 1H), 8.82 (m,1H), 8.7 (d, J=2.2 Hz, 1H), 8.6 (d, J=5.6 Hz, 1H), 8.36 (s, 1H), 8.17(m, 2H), 8.11 (m, 2H), 7.66 (d, J=5.6 Hz, 1H), 7.35 (m, 2H), 4.09 (s,3H), 3.19 (s, 3H), 2.35 (s, 3H). LC/MS (m/z) (M+1)⁺: 490.2.

N-(3-{4-[5-(2-Fluoro-ethoxy)-pyridin-3-yl]-pyrimidin-2-ylamino}-4-methyl-phenyl)-4-methanesulfonyl-benzamideD2

Final compound D2 is prepared from 17 with a similar procedure used forthe preparation of A1. ¹H NMR (400 MHz, d₆-DMSO) δ 10.5 (s, 1H), 9.04(s, 1H), 8.93 (d, J=1.6 Hz, 1H), 8.54 (d, J=5.2 Hz, 1H), 8.46 (d, J=2.8Hz, 1H), 8.16 (m, 3H), 8.07 (m, 3H), 7.5 (m, 2H), 7.24 (d, J=8.5 Hz,1H), 4.7 (d, J=47.8 Hz, 2H), 4.37 (d, J=30 Hz, 2H), 3.29 (s, 3H), 2.24(s, 3H). LC/MS (m/z) (M+1)⁺: 522.2.

Type E4-Methanesulfonyl-N-{4-methyl-3-[4-(5-methyl-pyridin-3-yl)-pyrimidin-2-ylamino]-phenyl}-benzamideE1

Final compound E1 is prepared from 21 with a similar procedure used forthe preparation of A1. LC/MS (m/z) (M+1)⁺: 474.2.

Type F3-(2-Fluoro-ethoxy)-4-methanesulfonyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamideF1

Compound 23 (0.04 mmol), CsHCO₃ (0.1 mmol) and 1-bromo-2-fluoro-ethane(0.04 mmol) are heated in dry acetonitrile (0.5 mL) at 150° C. in amicrowave oven for 10 min. Purification by preparative LC/MS affordsfinal compound F1. ¹H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 9.31 (s, 1H),9.01 (s, 1H), 8.73 (s, 1H), 8.55 (m, 2H), 8.10 (s, 1H), 7.95 (m, 1H),7.76 (s, 1H), 7.71 (m, 1H), 7.60 (m, 1H), 7.46 (m, 2H), 7.25 (d, J=8.0Hz, 1H), 4.92 (m, 1H), 4.80 (m, 1H), 4.62 (m, 1H), 4.55 (m, 1H), 3.32(s, 3H), 2.25 (s, 3H). LC/MS (m/z) (M+1)⁺: 522.2.

Similar procedures are used to prepare compounds F2-F8

Type GN-(3-(4-(isoquinolin-4-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3-(methylsulfonyl)benzamideG1

Final compound G1 is prepared from 27 with a similar procedure used forthe preparation of A1. LC/MS (m/z) (M+1)⁺: 510.2.

By repeating the procedures described in the above examples(intermediates and final compounds), using appropriate startingmaterials, the following compounds of Formula I, as identified in Table1, are obtained.

TABLE 1 Example # Structure MS [M + 1]⁺ A1

460.2 A2

460.1 A3

494.1 A4

440.2 A5

494.2 A6

474.2 A7

474.2 A8

504.2 A9

490.1 A10

544.2 B1

530.1 B2

546.2 C1

545.3 D1

490.2 D2

522.2 D3

530.2 D4

526.2 D5

476.1 E1

474.2 F1

522.2 F2

518.2 F3

490.2 F4

532.2 F5

530.2 F6

532.2 F7

558.1 F8

586.2 G1

510.2

Assays

Compounds of the present invention are assayed to measure their capacityto selectively inhibit the proliferation of wild type Ba/F3 cells andBa/F3 cells transformed with Tel c-kit kinase and Tel PDGFR fusedtyrosine kinases. In addition, compounds of the invention selectivelyinhibit SCF dependent proliferation in Mo7e cells. Further, compoundsare assayed to measure their capacity to inhibit Abl, ARG, BCR-Abl, BRK,EphB, Fms, Fyn, KDR, c-Kit, LCK, PDGF-R, b-Raf, c-Raf, SAPK2, Src, Tie2and TrkB kinases.

Proliferation Assay: BaF3 Library—Bright Glo Readout Protocol

Compounds are tested for their ability to inhibit the proliferation ofwt Ba/F3 cells and Ba/F3 cells transformed with Tel fused tyrosinekinases. Untransformed Ba/F3 cells are maintained in media containingrecombinant IL3. Cells are plated into 384 well TC plates at 5,000 cellsin 50 ul media per well and test compound at 0.06 nM to 10 μM is added.The cells are then incubated for 48 hours at 37° C., 5% CO₂. Afterincubating the cells, 25 μL of BRIGHT GLO® (Promega) is added to eachwell following manufacturer's instructions and the plates are read usingAnalyst GT—Luminescence mode—50000 integration time in RLU. IC₅₀ values,the concentration of compound required for 50% inhibition, aredetermined from a dose response curve.

Mo7e Assay

The compounds described herein are tested for inhibition of SCFdependent proliferation using Mo7e cells which endogenously expressc-kit in a 96 well format. Briefly, two-fold serially diluted testcompounds (Cmax=10 μM) are evaluated for their antiproliferativeactivity of Mo7e cells stimulated with human recombinant SCF. After 48hours of incubation at 37° C., cell viability is measured by using a MTTcolorimetric assay from Promega.

c-kit HTRF Protocol

An aliquot (5 μL) of a 2× concentration of c-kit enzyme mix 25 ng c-kit(5 ng/μL) and 2 μM of Biotin-EEEPQYEEIPIYLELLP-NH₂ peptide in kinasebuffer (20 mM Tris pH 7.5, 10 mM MgCl₂, 0.01% BSA, 0.1% Brij35, 1 mMDTT, 5% glycerol, 0.05 mM Na₃VO₄) is added to each well of a 384proxiplate (Packard). Each well of the last row of the proxiplate has 5μL of c-kit enzyme mix without c-kit to ascertain the background level.Compounds of the invention are added to each well and the plates areincubated for 30 minutes at room temperature. 2×ATP (40 μM) in kinasebuffer (5 μL) is added to each well and the plate is incubated at roomtemperature form 3 hours. Detection mix (50% KF, 40% kinase buffer, 10%EDTA, 1:100 diluted Mab PT66-K (cat#61T66KLB) and 1:100 dilutedStreptavidin-XL (cat#611SAXLB)0 (10 μL) is added to each well and theplates are further incubated for 1 to 2 hours at room temperature. TheHTRF signal is then read on a detector.

Human TG-HA-VSMC Proliferation Assay

Human TG-HA-VSMC cells (ATCC) are grown in DMEM supplemented with 10%FBS to 80-90% confluence prior to resuspending in DMEM supplemented with1% FBS and 30 ng/mL recombinant human PDGF-BB at 6e4 cells/mL. Cells arethen aliquoted into 384 well plates at 50 uL/well, incubated for 20 h at37° C., then treated with 0.5 uL of 100× compounds for 48 h at 37° C.After the treatment, 25 uL of CellTiter-Glo is added to each well for 15min, then the plates are read on the CLIPR (Molecular Devices).

PDGFRα & β Lance Assay Protocol

An aliquot (2.5 μL) of a 2× concentration of PDGFRβ peptide and ATP mix(4 μM biotin-βA-βA-βA-AEEEEYVFLEAKKK peptide, 20 μM ATP in assay buffer(20 mM Hepes, 54 mM MgCl₂, 0.01% BSA, 0.05% Tween-20, 1 mM DTT, 10%glycerol, 50 μM Na₃VO₄)) is added to each well of a 384 proxiplate(Packard). The plates are centrifuged and compounds of the invention (50nL) are added to each well via a pintool dispenser. To each well isadded (2.5 μL) of a 2× concentration of enzyme mix (PDGFRα at 4.5 ng/μL(cat# PV4117) or PDGFRβ at 1.5 ng/μL (cat# PV3591) in assay buffer) orassay buffer alone without PDGFRα/β enzyme. The plates are incubated for1.5 hours at room temperature. Detection mix (5 μL; 50% 1M KF, 40%kinase buffer, 10% EDTA, 1:100 diluted Mab PT66-K (cat#61T66KLB) and1:100 diluted Streptavidin-XL (cat#611SAXLB) is added to each well andthe proxiplate is incubated for 1 hour at room temperature beforereading the HTRF signal on a detector.

Ba/F3 FL FLT3 Proliferation Assay

The murine cell line used is the Ba/F3 murine pro-B cell line that overexpresses full length FLT3 construct. These cells are maintained in RPMI1640/10% fetal bovine serum (RPMI/FBS) supplemented with penicillin 50μg/mL, streptomycin 50 μg/mL and L-glutamine 200 mM with the addition ofmurine recombinant IL3. Ba/F3 full length FLT3 cells undergo IL3starvation for 16 hours and then plated into 384 well TC plates at 5,000cells in 25 uL media per well and test compound at 0.06 nM to 10 μM isadded. After the compound addition FLT3 ligand or IL3 for cytotoxicitycontrol are added in 25 ul media per well at the appropriateconcentrations. The cells are then incubated for 48 hours at 37° C., 5%CO₂. After incubating the cells, 25 μL of BRIGHT GLO® (Promega) is addedto each well following manufacturer's instructions and the plates areread using Analyst GT—Luminescence mode—50000 integration time in RLU.

Inhibition of Cellular BCR-Abl Dependent Proliferation (High ThroughputMethod)

The murine cell line used is the 32D hemopoietic progenitor cell linetransformed with BCR-Abl cDNA (32D-p210). These cells are maintained inRPMI/10% fetal calf serum (RPMI/FCS) supplemented with penicillin 50μg/mL, streptomycin 50 μg/mL and L-glutamine 200 mM. Untransformed 32Dcells are similarly maintained with the addition of 15% of WEHIconditioned medium as a source of IL3.

50 μL of a 32D or 32D-p210 cells suspension are plated in Greiner 384well microplates (black) at a density of 5000 cells per well. 50 nL oftest compound (1 mM in DMSO stock solution) is added to each well(STI571 is included as a positive control). The cells are incubated for72 hours at 37° C., 5% CO₂. 10 μL of a 60% Alamar Blue solution (Tekdiagnostics) is added to each well and the cells are incubated for anadditional 24 hours. The fluorescence intensity (Excitation at 530 nm,Emission at 580 nm) is quantified using the Acquest™ system (MolecularDevices).

Inhibition of Cellular BCR-Abl Dependent Proliferation

32D-p210 cells are plated into 96 well TC plates at a density of 15,000cells per well. 50 μL of two fold serial dilutions of the test compound(C_(max) is 40 μM) are added to each well (STI571 is included as apositive control). After incubating the cells for 48 hours at 37° C., 5%CO₂, 15 μL of MTT (Promega) is added to each well and the cells areincubated for an additional 5 hours. The optical density at 570 nm isquantified spectrophotometrically and IC₅₀ values, the concentration ofcompound required for 50% inhibition, determined from a dose responsecurve.

Effect on Cell Cycle Distribution

32D and 32D-p210 cells are plated into 6 well TC plates at 2.5×10⁶ cellsper well in 5 mL of medium and test compound at 1 or 10 μM is added(STI571 is included as a control). The cells are then incubated for 24or 48 hours at 37° C., 5% CO₂. 2 mL of cell suspension is washed withPBS, fixed in 70% EtOH for 1 hour and treated with PBS/EDTA/RNase A for30 minutes. Propidium iodide (Cf=10 μg/ml) is added and the fluorescenceintensity is quantified by flow cytometry on the FACScalibur™ system (BDBiosciences). Test compounds of the present invention demonstrate anapoptotic effect on the 32D-p210 cells but do not induce apoptosis inthe 32D parental cells.

Effect on Cellular BCR-Abl Autophosphorylation

BCR-Abl autophosphorylation is quantified with capture Elisa using ac-abl specific capture antibody and an antiphosphotyrosine antibody.32D-p210 cells are plated in 96 well TC plates at 2×10⁵ cells per wellin 50 μL of medium. 50 μL of two fold serial dilutions of test compounds(C_(max) is 10 μM) are added to each well (STI571 is included as apositive control). The cells are incubated for 90 minutes at 37° C., 5%CO₂. The cells are then treated for 1 hour on ice with 150 μL of lysisbuffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA and 1%NP-40) containing protease and phosphatase inhibitors. 50 μL of celllysate is added to 96 well optiplates previously coated with anti-ablspecific antibody and blocked. The plates are incubated for 4 hours at4° C. After washing with TBS-Tween 20 buffer, 50 μL ofalkaline-phosphatase conjugated anti-phosphotyrosine antibody is addedand the plate is further incubated overnight at 4° C. After washing withTBS-Tween 20 buffer, 90 μL of a luminescent substrate are added and theluminescence is quantified using the Acquest™ system (MolecularDevices). Test compounds of the invention that inhibit the proliferationof the BCR-Abl expressing cells, inhibit the cellular BCR-Ablautophosphorylation in a dose-dependent manner.

Effect on Proliferation of Cells Expressing Mutant Forms of Bcr-Abl

Compounds of the invention are tested for their antiproliferative effecton Ba/F3 cells expressing either wild type or the mutant forms ofBCR-Abl (G250E, E255V, T315I, F317L, M351T) that confers resistance ordiminished sensitivity to STI571. The antiproliferative effect of thesecompounds on the mutant-BCR-Abl expressing cells and on the nontransformed cells were tested at 10, 3.3, 1.1 and 0.37 μM as describedabove (in media lacking IL3). The IC₅₀ values of the compounds lackingtoxicity on the untransformed cells were determined from the doseresponse curves obtained as describe above.

FGFR3 (Enzymatic Assay)

Kinase activity assay with purified FGFR3 (Upstate) is carried out in afinal volume of 10 μL containing 0.25 μg/mL of enzyme in kinase buffer(30 mM Tris-HCl pH7.5, 15 mM MgCl₂, 4.5 mM MnCl₂, 15 μM Na₃VO₄ and 50μg/mL BSA), and substrates (5 μg/mL biotin-poly-EY(Glu, Tyr) (CIS-US,Inc.) and 3 μM ATP). Two solutions are made: the first solution of 5 μLcontains the FGFR3 enzyme in kinase buffer was first dispensed into384-format ProxiPlate® (Perkin-Elmer) followed by adding 50 nL ofcompounds dissolved in DMSO, then 5 μL of second solution contains thesubstrate (poly-EY) and ATP in kinase buffer was added to each wells.The reactions are incubated at room temperature for one hour, stopped byadding 10 μL of HTRF detection mixture, which contains 30 mM Tris-HClpH7.5, 0.5 M KF, 50 mM ETDA, 0.2 mg/mL BSA, 15 μg/mL streptavidin-XL665(CIS-US, Inc.) and 150 ng/mL cryptate conjugated anti-phosphotyrosineantibody (CIS-US, Inc.). After one hour of room temperature incubationto allow for streptavidin-biotin interaction, time resolved florescentsignals are read on Analyst GT (Molecular Devices Corp.). IC₅₀ valuesare calculated by linear regression analysis of the percentageinhibition of each compound at 12 concentrations (1:3 dilution from 50nM to 0.28 nM). In this assay, compounds of the invention have an IC₅₀in the range of 10 nM to 2 μM.

FGFR3 (Cellular Assay)

Compounds of the invention are tested for their ability to inhibittransformed Ba/F3-TEL-FGFR3 cells proliferation, which is depended onFGFR3 cellular kinase activity. Ba/F3-TEL-FGFR3 are cultured up to800,000 cells/mL in suspension, with RPMI 1640 supplemented with 10%fetal bovine serum as the culture medium. Cells are dispensed into384-well format plate at 5000 cell/well in 50 μL culture medium.Compounds of the invention are dissolved and diluted in dimethylsufoxide(DMSO). Twelve points 1:3 serial dilutions are made into DMSO to createconcentrations gradient ranging typically from 10 mM to 0.05 μM. Cellsare added with 50 nL of diluted compounds and incubated for 48 hours incell culture incubator. AlamarBlue® (TREK Diagnostic Systems), which canbe used to monitor the reducing environment created by proliferatingcells, are added to cells at final concentration of 10%. After anadditional four hours of incubation in a 37° C. cell culture incubator,fluorescence signals from reduced AlamarBlue® (Excitation at 530 nm,Emission at 580 nm) are quantified on Analyst GT (Molecular DevicesCorp.). IC₅₀ values are calculated by linear regression analysis of thepercentage inhibition of each compound at 12 concentrations.

b-Raf—Enzymatic Assay

Compounds of the invention are tested for their ability to inhibit theactivity of b-Raf. The assay is carried out in 384-well MaxiSorp plates(NUNC) with black walls and clear bottom. The substrate, IκBα is dilutedin DPBS (1:750) and 15 μL is added to each well. The plates areincubated at 4° C. overnight and washed 3 times with TBST (25 mM Tris,pH 8.0, 150 mM NaCl and 0.05% Tween-20) using the EMBLA plate washer.Plates are blocked by Superblock (15 μL/well) for 3 hours at roomtemperature, washed 3 times with TBST and pat-dried. Assay buffercontaining 20 μM ATP (10 μL) is added to each well followed by 100 nL or500 nL of compound. B-Raf is diluted in the assay buffer (1 μL into 25μL) and 10 μL of diluted b-Raf is added to each well (0.4 μg/well). Theplates are incubated at room temperature for 2.5 hours. The kinasereaction is stopped by washing the plates 6 times with TBST. Phosph-IκBα(Ser32/36) antibody is diluted in Superblock (1:10,000) and 15 μL isadded to each well. The plates are incubated at 4° C. overnight andwashed 6 times with TBST. AP-conjugated goat-anti-mouse IgG is dilutedin Superblock (1:1,500) and 15 μL is added to each well. Plates areincubated at room temperature for 1 hour and washed 6 times with TBST.15 μL of fluorescent Attophos AP substrate (Promega) is added to eachwell and plates are incubated at room temperature for 15 minutes. Platesare read on Acquest or Analyst GT using a Fluorescence Intensity Program(Excitation 455 nm, Emission 580 nm).

b-Raf—Cellular Assay

Compounds of the invention are tested in A375 cells for their ability toinhibit phosphorylation of MEK. A375 cell line (ATCC) is derived from ahuman melanoma patient and it has a V599E mutation on the B-Raf gene.The levels of phosphorylated MEK are elevated due to the mutation ofB-Raf. Sub-confluent to confluent A375 cells are incubated withcompounds for 2 hours at 37° C. in serum free medium. Cells are thenwashed once with cold PBS and lysed with the lysis buffer containing 1%Triton X100. After centrifugation, the supernatants are subjected toSDS-PAGE, and then transferred to nitrocellulose membranes. Themembranes are then subjected to western blotting with anti-phospho-MEKantibody (ser217/221) (Cell Signaling). The amount of phosphorylated MEKis monitored by the density of phospho-MEK bands on the nitrocellulosemembranes.

Upstate KinaseProfiler™—Radio-Enzymatic Filter Binding Assay

Compounds of the invention are assessed for their ability to inhibitindividual members of the kinase panel. The compounds are tested induplicates at a final concentration of 10 μM following this genericprotocol. Note that the kinase buffer composition and the substratesvary for the different kinases included in the “Upstate KinaseProfiler™”panel. Kinase buffer (2.5 μL, 10×-containing MnCl₂ when required),active kinase (0.001-0.01 Units; 2.5 μL), specific or Poly(Glu-4-Tyr)peptide (5-500 μM or 0.01 mg/ml) in kinase buffer and kinase buffer (50μM; 5 μL) are mixed in an eppendorf on ice. A Mg/ATP mix (10 μL; 67.5(or 33.75) mM MgCl₂, 450 (or 225) μM ATP and 1 μCi/μl [γ-³²P]-ATP (3000Ci/mmol)) is added and the reaction is incubated at about 30° C. forabout 10 minutes. The reaction mixture is spotted (20 μL) onto a 2 cm×2cm P81 (phosphocellulose, for positively charged peptide substrates) orWhatman No. 1 (for Poly (Glu-4-Tyr) peptide substrate) paper square. Theassay squares are washed 4 times, for 5 minutes each, with 0.75%phosphoric acid and washed once with acetone for 5 minutes. The assaysquares are transferred to a scintillation vial, 5 ml scintillationcocktail are added and ³²P incorporation (cpm) to the peptide substrateis quantified with a Beckman scintillation counter. Percentageinhibition is calculated for each reaction.

Compounds of Formula I, in free form or in pharmaceutically acceptablesalt form, exhibit valuable pharmacological properties, for example, asindicated by the in vitro tests described in this application.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A compound of Formula I:

in which L is selected from —NHC(O)—, —NHC(O)N— and —C(O)NH—; R₁, R_(2a)and R_(2b) are each independently selected from hydrogen, hydroxy,C₃₋₈heterocycloalkyl, C₁₋₄-alkyl, C₁₋₄-alkoxy,halo-substituted-C₁₋₄alkoxy, halo-substituted-C₁₋₄alkyl, —NR₁₀R₁₁,—OX₁R₈; wherein X₁ is selected from a bond and C₁₋₄alkylene; R₈ isC₃₋₁₂cycloalkyl; or R₁ and R_(2a) or R₁ and R_(2b) together with thecarbon atoms to which R₁ and R_(2a) or R_(2b) are attached form phenyl;R₁₀ and R₁₁ are independently selected from hydrogen, C₁₋₄-alkyl,C₁₋₄-alkoxy, halo-substituted-C₁₋₄alkoxy, halo-substituted-C₁₋₄-alkyl,C₃₋₈heterocycloalkyl, C₁₋₁₀heteroaryl; or R₁₀ and R₁₁ together with thenitrogen to which R₁₀ and R₁₁ are both attached formC₃₋₈heterocycloalkyl or C₁₋₁₀heteroaryl; R₃, R₄, R₅, R₆ and R₇ areindependently selected from hydrogen, halo, cyano, C₁₋₆alkyl,C₁₋₆alkoxy, halo-substituted-C₁₋₆alkoxy, C₃₋₈heterocycloalkyl, —OX₂R₉,—S(O)₀₋₂R₉ and —NR₁₂R₁₃; wherein X₂ is selected from a bond andC₁₋₄alkylene; and each R₉ is independently selected from hydrogen,C₁₋₆alkyl, C₁₋₆alkoxy, C₃₋₁₂cycloalkyl, and C₃₋₈heterocycloalkyl;wherein said cycloalkyl or heterocycloalkyl of R₉ is optionallysubstituted with 1 to 3 radicals independently selected from C₁₋₆alkyl,C₁₋₆alkoxy, halo-substituted-C₁₋₆alkoxy, halo-substituted-C₁₋₆alkyl and—NR₁₂R₁₃; wherein R₁₂ and R₁₃ are independently selected from hydrogenand C₁₋₆alkyl; or R₄ and R₅ together with the carbon atoms to which R₄and R₅ are attached form C₃₋₈heteroaryl; with the proviso that at leastone of R₃, R₄, R₅, R₆ or R₇ has a sulfur directly linked to the phenylring; and the pharmaceutically acceptable salts thereof.
 2. The compoundof claim 1 in which: L is selected from —NHC(O)— and —C(O)NH—; R₁,R_(2a) and R_(2b) are independently selected from hydrogen, hydroxy,C₃₋₈heterocycloalkyl, C₁₋₄alkyl, C₁₋₄alkoxy,halo-substituted-C₁₋₄alkoxy, halo-substituted-C₁₋₄alkyl, —NR₁₀R₁₁,—OX₁R₈; wherein X₁ is selected from a bond and C₁₋₄alkylene; R₈ isC₃₋₁₂cycloalkyl; or R₁ and R₂ together with the carbon atoms to which R₁and R_(2a) are attached form phenyl; R₁₀ and R₁₁ are independentlyselected from hydrogen, C₁₋₄alkyl, C₁₋₄alkoxy,halo-substituted-C₁₋₄alkoxy, halo-substituted-C₁₋₄alkyl,C₃₋₈heterocycloalkyl, C₁₋₁₀heteroaryl; or R₁₀ and R₁₁ together with thenitrogen to which R₁₀ and R₁₁ are both attached formC₃₋₈heterocycloalkyl or C₁₋₁₀heteroaryl; R₃ and R₇ are independentlyselected from hydrogen and halo; R₄ and R₆ are independently selectedfrom hydrogen, C₁₋₆alkyl, C₁₋₆alkoxy, halo-substituted-C₁₋₆alkoxy,—S(O)₀₋₂R₆; and R₅ is selected from halo, C₁₋₆alkyl, C₁₋₆alkoxy,halo-substituted-C₁₋₆alkoxy, C₃₋₈heterocycloalkyl, —OX₂R₉ and—S(O)₀₋₂R₉; wherein X₂ is selected from a bond and C₁₋₄alkylene; andeach R₉ is independently selected from hydrogen, C₁₋₆alkyl,C₃₋₁₂cycloalkyl and C₃₋₈heterocycloalkyl; wherein said cycloalkyl orheterocycloalkyl of R₉ is optionally substituted with 1 to 3 radicalsindependently selected from C₁₋₆alkyl and halo-substituted-C₁₋₆alkyl; orR₄ and R₅ together with the carbon atoms to which R₄ and R₅ are attachedform C₃₋₈heteroaryl, with the proviso that at least one of R₃, R₄, R₅,R₆ or R₇ has a sulfur directly linked to the phenyl ring.
 3. Thecompound of claim 2 in which R₁ is selected from hydrogen, hydroxy,pyrrolidinyl, morpholino, methoxy, difluoro-methoxy, 2-fluoro-ethoxy andmethyl; or R₁ and R_(2a) together with the carbon atoms to which R₁ andR_(2a) are attached form phenyl.
 4. The compound of claim 3 in which R₄and R₆ are independently selected from hydrogen, methyl-sulfonyl,methyl, 2-fluoro-ethoxy, methyl-piperazinyl-sulfonyl, propoxy,isobutoxy, 2,2,2-trifluoroethoxy, 2,3-difluoro-2-(fluoromethyl)propoxy,butoxy and cyclopropyl-methoxy; R₅ is selected from hydrogen, halo,methyl-sulfonyl, methyl, methoxy, ethoxy and morpholino; or R₄ and R₅together with the carbon atoms to which R₄ and R₅ are attached formthiazolyl.
 5. The compound of claim 1 selected from:N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;4-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzo[d]thiazole-6-carboxamide;2-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;3-methyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;4-methyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;4-ethoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(5-morpholinopyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(5-(pyrrolidin-1-yl)pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)-4-morpholinobenzamide;N-(3-(4-(5-methoxypyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;N-(3-(4-(5-(2-fluoroethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;N-(3-(4-(5-(cyclopropylmethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(5-methylpyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;3-(2-fluoroethoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)-3-propoxybenzamide;3-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;3-butoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;3-(cyclopropylmethoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(3-(4-(isoquinolin-4-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3-(methylsulfonyl)benzamide;4-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide;N-(3-(4-(5-(difluoromethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(4-methylpiperazin-1-ylsulfonyl)benzamide;N-(3-(4-(5-hydroxypyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide;3-isobutoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide;N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)-3-(2,2,2-trifluoroethoxy)benzamide;and3-(2,3-difluoro-2-(fluoromethyl)propoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide.6. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound of claim 1 in combination with a pharmaceuticallyacceptable excipient.
 7. The pharmaceutical composition of claim 6,wherein the pharmaceutically acceptable excipient is suitable forparenteral administration.
 8. The pharmaceutical composition of claim 6,wherein the pharmaceutically acceptable excipient is suitable for oraladministration.